Designing Novel Protein Assemblies as Rigid Symmetric Scaffolds for Cryo-EM Imaging

摘要 低温电子显微镜(CERO-EM)正在经历非凡的技术革命，使其 有可能在原子细节上成像大型蛋白质组件和核酸复合体。然而，关键的是， 这些方法主要适用于大型组件(特别是对称组件)，而较小的组件 蛋白质(例如小于50 kDa)低于当前的技术限制。考虑到平均细胞 蛋白质比这更小，迫切需要新的分子策略来充分实现 低温EM的转化潜力。 长期以来，人们一直认为，冷冻-EM可以显示出更小、更典型大小的蛋白质 如果它们可以严格排列在更大的分子支架上，以提供质量和更高的对比度，以及 理想的情况是，以一种高度对称的方式。这一目标在很大程度上难以实现。两把钥匙 挑战在于(1)如何以一种刚性的方式将蛋白质连接到支架上，从而使重要的成像 优势可以被利用，以及(2)如何开发不需要 对每个新目标进行不可预测的分子工程和优化以及费力的EM测试 有待研究的分子。 这项技术开发计划回答了开发低温EM支架的挑战 是对称的、模块化的和刚性的，通过应用我们团队和合作者的新方法来设计 精确定义和高度对称的蛋白质组件。设计的蛋白质笼提供了对称的 用于外部附件的核心。对于这些对称的核心，我们在基因上融合了被称为DARPins的蛋白质， 它们已被开发为与基于不同蛋白质的特异和刚性结合的简便系统 关于它们的环路序列的实验室进化。重要的是，DARPin和 通过使用连续的阿尔法螺旋融合方法，设计的对称核心变得相对刚性，以及 早些时候介绍了设计新型蛋白质笼的想法。 在前期工作中，我们通过冷冻对第一代模块化支架候选之一进行了评估。 嗯。当单独成像时(笼形核心加上DARPin)，核心被解析为3.1？，并且DARPin被保持 足够坚硬，可以看到中等分辨率(大约3.5到5.5？)。在新的初步数据中，第一个脚手架 候选人已被成像与第一个货物蛋白，绿色荧光蛋白，结合。在那里，DARPIN有点僵化 与自由形式相比，虽然绑定的GFP本身在总体分辨率为4.7ä的情况下可视，但新的 小分子蛋白质冷冻-EM的基准。这些实验展示了最有希望和最模块化的 到目前为止用于成像小蛋白的路线，同时也显示了需要进一步设计步骤才能达到 近原子分辨率。这项领先的工作将设计、评估和完善一套新型的冷冻-EM支架 基于设计的蛋白质笼，对结构生物学和医学有重大影响。

SUMMARY Cryo-electron microscopy (cyro-EM) is undergoing extraordinary revolutions in technology, making it possible to image large protein assemblies and nucleic acid complexes in atomic detail. Critically however, the methods are mainly applicable to large assemblies (especially those that are symmetric), while smaller proteins (e.g. smaller than 50 kDa) are below current technological limits. Given that the average cellular protein is smaller than that, new molecular strategies are needed badly in order to fully realize the transformational potential of cryo-EM. It has long been recognized that smaller proteins of more typical size could be visualized by cryo-EM if they could be arrayed rigidly on a larger molecular scaffold to provide mass and higher contrast, and ideally in a way that would confer a high degree of symmetry. This goal has been largely elusive. Two key challenges have been (1) how to attach proteins to scaffolds in a rigid way so that important imaging advantages could be exploited, and (2) how to develop a modular system that would not require unpredictable molecular engineering and optimization and laborious EM testing for every new target molecule to be studied. This Technology Development proposal answers the challenge of developing cryo-EM scaffolds that are symmetric, modular, and rigid, by applying new methods from our group and collaborators for designing precisely defined and highly symmetric protein assemblies. Designed protein cages provide a symmetric core for external attachments. To these symmetric cores, we genetically fuse proteins known as DARPins, which have been developed as facile systems for binding specifically and rigidly to diverse proteins based on laboratory evolution of their loop sequences. Importantly, the connection between the DARPin and the designed symmetric core is made relatively rigid by using a continuous alpha helical fusion approach, an idea introduced earlier for designing novel protein cages. In preliminary work we evaluated one of the first-generation modular scaffold candidates by cryo- EM. When imaged by itself (cage core plus DARPin) the core is resolved to 3.1Å and the DARPin is held rigidly enough to see at medium resolution (about 3.5 to 5.5 Å). In new preliminary data the first scaffold candidate has been imaged with a first cargo protein, GFP, bound. There the DARPin is somewhat rigidified compared to the free form, while the bound GFP itself is visualized at 4.7 Å resolution overall, a new benchmark for cryo-EM of small proteins. These experiments lay out the most promising and most modular route so far for imaging small proteins, while also showing what further design steps are required to reach near-atomic resolution. This leading work will design, evaluate and perfect a novel set of cryo-EM scaffolds based on designed protein cages, with major impacts on structural biology and medicine.